This research was supported by the S-Innovation project of the Japan Science and technology Agency (JST), the Graduate Program for Leaders in Life Innovation (GPLLI) of the University of Tokyo, and the Research Fellowship for Young Scientists of Japan Society for the Promotion of Science. We thank nac Image Technology Inc. for taking movies using a hi-speed camera.

1. Hazırlanması Malzemeler <ol><li> 10 mM HEPES tamponu hazırlayın. RT&#39;de 7.0 pH ayarlayın ve% 0,9 ile NaCl ekleyin. </li><li> 1,1 hazırlanan HEPES-tamponlu tuzlu su karıştırılarak% 5 aljinat çözeltisi ve 10 mM EDTA solüsyonu hazırlayın. Oda sıcaklığında 7.0 pH ayarlayın. </li><li> 121 ° C&#39;de 20 dakika için reaktifler (1.1, 1.2) Otoklav. </li><li> DMEM,% 10 ES nitelikli FBS ve 90, mitomisin C, 10 ug / ml ihtiva eden içinde kuluçkalama ile besleyici hücreler olarak kabul edilir 2.0 x 10 6 fare embriyonik akciğer fibroblast (MEF) hücreleri, - 1.2 katmanlı jelatin kaplı 60 mm&#39;lik yemekler hazırlamak - 120 dk. </li><li> (ES nitelikli FBS% 20, 2-merkaptoetanol, temel olmayan amino asit ve antibiyotik 50 uM içeren 5 ml DMEM yüksek glikoz mevcudiyetinde besleyici hücreler ile tabağına hücreleri (IPS) fare uyarılmış pluripotent kök bakımı Penisilin G Sodyum, streptomisin sülfat, 100 ug / ml, 100 birim / ml ve amphoteri 250 ng / mlcin B). </li><li> Tohum 4 × 10 5 iPS bir jelatin kaplı 60 mm çanak hücreleri ve CO 2 37 ° C&#39;de inkübe ve% 5. Inkübasyon sırasında kültür ortamı her gün değiştirin. 3 sonra, - kültür 4 gün, 37 ° C&#39;de% 0.05 tripsin,% 0.02 EDTA ihtiva eden 500 ul 1 dakika% 5 CO2 hücreleri trypsinize. Bundan sonra, kültür çanak on kere dokunarak hücreleri ayırmak. </li><li> 3 dakika boyunca 160 x g&#39;de santrifüj hücre süspansiyonu (üç kez 1.000 ul mikropipet ile pipetleme tek hücre içine ayrışma fare iPS hücrelerinin takiben) FBS ve antibiyotik ES-nitelikli% 10 ile DMEM 2.5 ml ekleyin ve süpernatant kaldırmak ve hücre sayımı . Hücre sayımı sonrasında, bir jelatin-kaplı tabak üzerinde toplanan hücreler kalkarlar ve MEF hücrelerinden iPS hücrelerini izole etmek için 30 dakika boyunca inkübe edin. Hücrelerin sayılmasından sonra (genellikle 1-3 x 10 6 hücre / çanak toplanabilir), 4 x 10 5 iPS hücrelerini reseedçanak. </li></ol> Aljinat Hidrojel Kapsüller içine 2. Hücre Encapsulation <ol><li> 1.5 ve 1.6 de tarif edilen ayni yöntem vasıtasıyla hücreleri toplamak. 3 dakika boyunca 160 x g&#39;de santrifüj sonra, üç kez HEPES tamponlu salin ile iPS hücresi pelet yıkayın. HEPES-tamponlu tuzlu su içinde hücrelerin yeniden süspansiyona sonra, başka şekilde memeyi tıkayabilecek büyük topaklar ortadan kaldırmak üzere bir 40 um&#39;lik bir hücre süzgecinden hücre süspansiyonu filtre edin. </li><li> HEPES-tamponlu tuzlu su içinde hücre süspansiyonu 2 ml ve% 5 Alg-Na çözeltisi 3 ml karıştırın. Son olarak% 3 Alg-Na çözeltisi içinde hücre 5 ml süspansiyon elde edilir. Hücre yoğunluğu tercihen en fazla 10 6 hücre / ml &#39;dir. </li><li> 5 ml enjektöre hücre süspansiyonu topladıktan sonra, şırınga üzerindeki bir eş eksenli meme (Şekil 1A, B) yüklemek ve şırınga pompası bunları ayarlayın. Emit N2 gazı akışı (düşük 1L / dakika daha) eş-eksenli, memenin dış iğne yoluyla veİç memeden hücre süspansiyonu sınırdışı. </li><li> % 0.5 CaCI2 çözeltisi 250 ml damlacıklar toplamak ve 10 bekle - 60-90 rpm&#39;de karıştırılırken katılaşma işlemi için 20 dakika. </li></ol> Aljinat Capsule&#39;lerden 3. Tedavi (isteğe bağlı) <ol><li> % 0.05 Alg-Ca-kapsüller inkübe poli-L-lisin (PLL), 37 ° C&#39;de, 5 dakika için çözelti ve% 5 (w / v) CO2. </li><li> HEPES-tamponlu tuzlu su ile yıkama işleminden sonra kapsül, DMEM yüzeyleri üzerindeki elektrik yükü nötralize etmek için% 10 FBS ihtiva eden inkübe. Kaplanmış kapsüller (Şekil 1C) olarak kullanmaktadır. </li><li> 5 dakika boyunca 10 mM EDTA ihtiva eden HEPES-tamponlu tuzlu su içinde kapsül inkübe edin. EDTA ile muamele edilmiş kapsüller boş kapsüller (Şekil 1C) olarak kullanılır. </li></ol> Aljinat Kapsüllerinde 4. Kültür ve toplayın Hücreler <ol><li> 37 ° C ve% 5 CO2 ile çalkalanarak 75 rpm&#39;de kapsüllenmiş hücreleri inkübe10 gün boyunca uygulanır. </li><li> Toplamak ve 10 dakika boyunca CO2, 37 ° C&#39;de, 10 mM EDTA içinde kapsül inkübe ve% 5. 3 dakika boyunca 1000 x g&#39;da santrifüjleme ile PLL muamelesi olmadan aljinat kapsüllerinden hücreleri toplamak. </li><li> 5 dakika boyunca 5,000 × g - aljinat kapsüller PLL ile tedavi ise, yukarı ve aşağı pipetleme on kat yaklaşık 1.000 bir şırınga ve santrifüj bunu bağlı bir iğne ile tarafından Alg-PLL membran bölünürler. İğne çapı kapsül boyutu ve 25 g (260 um) iğnelerin, bu deneyde kullanılan daha düşük olmalıdır (600 um) </li><li> Eğer hücrelerden mRNA örnekleri toplamak istiyorsanız santrifüj sonra, kırık Alg-PLL zarlardan kesinlikle hücre pelet toplamak. Kalan Alg-PLL membranlar muhtemelen mRNA Saflaştırma önler. </li></ol>

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The authors have nothing to disclose.

<html> Kapsülleme kültürü doğrudan süspansiyon kültürleri ile karşılaştırılabilir. Süspansiyon kültürü kapsülleme yöntemleri daha pluripotent kök hücrelerin büyük miktarlarda elde etmek için basit bir yöntemdir. Bununla birlikte, süspansiyon kültüründe hücrelerin toplanmasını kontrol etmek de zordur. Kapsülleme yönteminde, hücre-çekiş kapsüller sınırlıdır ve bu nedenle de iyi bir şekilde kontrol edilebilir. Önceki bir yayın büyük hücre kümeleri serbest süspansiyon kültü...

Induced pluripotent stem cells (iPS cells) are currently the source of intense research due to their role in regenerative medicine. However, huge amounts of cells are required for tissue regeneration. For instance approximately one billion pancreatic cells required for a type 1 diabetic patient1. However, conventional dish culture is only able to obtain 1 &#215; 105 cells/cm2, thus requiring 1 m2 of culture area to obtain enough stem cell-derived pancreatic cells to treat a type 1 diabetic patient. The development of a system for the mass-culture of pluripotent stem cells, such as microcarrier2 and suspension culture is therefore required for regenerative medicine. Suspension culture represents a promising method of mass culture but controlling the aggregation of cells is challenging in direct suspension cultures of human iPS cells3. Indeed, suspended cells are exposed to shear stress, which causes cell damage3 or differentiation4.
Research into hydrogel-based encapsulation has been conducted to solve problems associated with suspension culture. In hydrogel capsules, cells are protected from the flow of the medium. Previous reports have documented the use of various types of hydrogel, including agarose5, PEG6, and alginate (Alg), for cellular encapsulation. Alg-Ca hydrogel is one of the most useful hydrogels for cell encapsulation because Alg&#8722;Ca hydrogel is formed immediately after dropping alginate solution into a CaCl2 solution and is also readily digested by enzymes or chelating reagents. 
Here, we have established a stable alginate encapsulation process for iPS cells using a co-axial nozzle. By using N2 gas flow for forming droplets, it is possible to encapsulate cells into uniform capsules without the need for other reagents such as oil. In this method, the flow rate of N2 and concentration of both CaCl2 and alginate are the major operating conditions affecting the size, shape, and uniformity of capsules. This report demonstrates the optimization of these operating conditions through the use of a hi-speed camera and a microscope.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Mouse embryo fibroblast</td>
			<td>Cell Biolabs</td>
			<td>SNL 76/7</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse induced pluripotent stem cell</td>
			<td>RIKEN Bio resorce centre</td>
			<td>iPS-MEF-Ng-20D-17</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high-glucose</td>
			<td>GIBCO</td>
			<td>11995</td>
			<td></td>
		</tr>
		<tr>
			<td>ES qualified&#160; FBS</td>
			<td>GIBCO</td>
			<td>16141079</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibacterial Antibiotics</td>
			<td>GIBCO</td>
			<td>15240</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential Amino Acid</td>
			<td>GIBCO</td>
			<td>11140</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>GIBCO</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>ESGRO Leukemia Inhibitory Factor</td>
			<td>Merck Millipore</td>
			<td>ESG1107</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>GIBCO</td>
			<td>25300</td>
			<td></td>
		</tr>
		<tr>
			<td>26G/16G needle</td>
			<td>Hoshiseido</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Syringe</td>
			<td>TERUMO</td>
			<td>SS-10ESZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium&#160; Chloride</td>
			<td>Wako</td>
			<td>191-01665</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>SIGMA</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Alginate</td>
			<td>Wako</td>
			<td>194-09955</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Wako</td>
			<td>039-00475</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine (Mw=15,000-30,000)</td>
			<td>SIGMA</td>
			<td>P7890</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>DOJINDO</td>
			<td>345-01865</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylinge pump</td>
			<td>AS ONE</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>IX71</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica</td>
			<td>DM IRB</td>
			<td></td>
		</tr>
		<tr>
			<td>Hispeed camera</td>
			<td>nac image technology</td>
			<td>Memrecam HX-3</td>
			<td></td>
		</tr>
	</tbody>
</table>

alginate encapsulation of pluripotent stem cells using a co-axial nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

Protokole 2.5 anda, sınırdışı aljinat çözüm sınırdışı hemen sonra küresel bir şekil (- H Şekil 2A) oluşturur. Süspansiyon 1 L / dak daha düşük bir N2 akış oranı ile dışarı atılır ise, damlacıkların boyutunun düzgün (Şekil 2I) &#39;dir. N2 akışı 1 L / dak daha yüksek olan, ancak, damlacık (Şekil 2G, beyaz ok) ayırır ve damlacıkların boyutu, heterojen (Şekil 2J) olur. Bu göz önüne alın...

Watch this Scientific Journal Video about Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle at JoVE.com

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

Bir Koaksiyel Memesini kullanma Pluripotent Kök Hücre Aljinat Encapsulation

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

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Developmental Biology

12.5K Görüntüleme.  The University of Tokyo. We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.